Increased bacterial CoA and acetyl-CoA pools

ABSTRACT

Methods of increasing the cellular pool of A-CoA and thus driving the metabolic pathways in the direction of A-CoA containing metabolites by overexpressing rate limiting enzymes in A-CoA synthesis. Methods of increasing intracellular levels of CoA and A-CoA through genetic engineering of bacterial strains in conjunction with supplementation with precursor molecules.

PRIOR RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/457,093, filed Mar. 24, 2003 and U.S. ProvisionalApplication No. 60/457,635, filed Mar. 26, 2003.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] The present invention has been developed with funds from theNational Science Foundation and United States Department of Agriculture.Therefore, the United States Government may have certain rights in theinvention.

REFERENCE TO APPENDIX

[0003] A Sequence Listing, including SEQ ID NO: 1 and 2, is submittedwith this application.

FIELD OF THE INVENTION

[0004] The invention relates to methods of increasing intracellularlevels or flux of CoA and A-CoA through genetic engineering of bacterialstrains in conjunction with supplementation with precursor molecules.The invention further relates to methods of increasing the cellular poolor flux of A-CoA and thus driving the metabolic pathways in thedirection of A-CoA containing metabolites and A-CoA derivatives.

BACKGROUND OF THE INVENTION

[0005] Coenzyme A (CoA) and its thioester derivative Acetyl CoA (A-CoA)are essential intermediates in numerous biosynthetic and energy yieldingmetabolic pathways as well as regulators of several key metabolicreactions. A-CoA is an important intracellular metabolite in centralcarbon metabolism and is a precursor in the enzymatic synthesis of manyuseful compounds. A-CoA is formed during the enzymatic oxidation ofpyruvate or fatty acids, and from free acetate in the presence of theenzyme acetyl-CoA synthase. There are several key rate limiting steps inthe biosynthesis of A-CoA. The overexpression of the enzymes catalyzingthese rate limiting steps increases the intracellular levels of A-CoA.The A-CoA node serves as a connecting point at which several metabolicpathways intersect. Enhancing the A-CoA flux, i.e., the amount of A-CoAgenerated in a given time, through the A-CoA node is a useful strategyfor increasing the production of compounds that require A-CoA for theirbiosynthesis.

[0006] CoA and A-CoA are precursors to many industrially usefulcompounds. A-CoA is also a substrates in alcohol acetyl transferasereactions that produce various acetate esters. In addition, A-CoA andits condensation product acetoacetyl-CoA are involved in the biologicalproduction of various polyhydroxybutyrates (PHBs). A-CoA can becarboxylated to malonyl-CoA and subsequently enter pathways toisoprenoid and terpenoid compounds through mevalonate. In sum, enhancingthe intracellular pools/flux of A-CoA has implications in improving theproduction of the useful compounds derived from A-CoA.

[0007] Existing methodologies focus on the engineering of metabolicpathways by overexpressing enzymes that are directly involved in theproduction of a target compound. The invention claimed and describedherein differs from existing methodologies in that in the presentinvention, cellular metabolism is altered to increase glycolytic fluxand to direct this increased flux towards the production of precursormolecules such as A-CoA. The increased production of A-CoA in turnincreases the production of target compounds such as esters, PHBs andpolyketides.

[0008] Metabolic engineering has the potential to considerably improveprocess productivity by manipulating the throughput of metabolicpathways. Most current metabolic engineering studies focus onmanipulating enzyme levels through the amplification, addition, ordeletion of a particular pathway. However, cofactors play an essentialrole in a large number of biochemical reactions and their manipulationhas the potential to be used, as an additional tool to achieve desiredmetabolic engineering goals. In addition, cofactor manipulation may alsoprovide an additional means to study cellular metabolism, in particularthe interplay between cofactor levels/fluxes and metabolic fluxes.

SUMMARY OF THE INVENTION

[0009] An aspect of the invention provides a method for increasing thelevels of CoA or A-CoA in an E. coli strain through the geneticmanipulation of the strain. Another aspect of the invention provides amicroorganism with increased intracellular levels of CoA or A-CoA.

[0010] An aspect of the invention provides a method for manipulating themetabolism of a cell, comprising expression at elevated levels of one ormore enzymes involved in A-CoA metabolism, wherein the cell displaysincreased flux through the A-CoA node.

[0011] A further aspect of the invention provides a microorganism whichexpresses one or more enzymes involved in A-CoA metabolism at elevatedlevels, wherein said microorganism displays increased flux through theA-CoA node.

[0012] An aspect of the invention provides a method of producing isoamylacetate in a cell comprising expression at elevated levels of one ormore enzymes involved in A-CoA metabolism, wherein the cell displaysincreased flux through the A-CoA node

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings which are incorporated in andconstitute a part of this specification exemplify the invention andtogether with the description, serve to explain the principles of theinvention:

[0014]FIG. 1 illustrates the metabolic pathway at the A-CoA node;

[0015]FIG. 2 illustrates the involvement of pantothenate kinase in thebiosynthesis of CoA;

[0016]FIG. 3 illustrates a plasmid construct used for the overexpressionof pantothenate kinase;

[0017]FIG. 4 illustrates metabolite concentrations of acetate, glucoseand succinate in bacterial strains overexpressing pantothenate kinase;

[0018]FIG. 5 illustrates intracellular CoA and A-CoA levels of steadystate chemostat cultures;

[0019]FIG. 6 illustrates intracellular CoA and A-CoA levels in bacterialstrains overexpressing pantothenate kinase;

[0020]FIG. 7 illustrates levels of CoA, A-CoA and isoamyl acetate inbacterial strains overexpressing pantothenate kinase in the presence ofpantothenate supplement;

[0021]FIG. 8 illustrates the glucose uptake rate of steady statechemostat cultures;

[0022]FIG. 9 illustrates the acetate production rate of steady statechemostat cultures;

[0023]FIG. 10 illustrates isoamyl acetate concentrations in the strainstested; and

[0024]FIG. 11 illustrates pyruvic acid concentrations in the strainstested.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0025] Reference will now be made in detail to embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

[0026] An application of this invention can be to increase theproduction of esters, PHBs and polyketides. Coenzyme A (CoA) and A-CoAare precursors to fatty acid biosynthesis. Hence with the manipulationof CoA and A-CoA, fatty acid biosynthesis can potentially be altered.

[0027] Esters are an important class of chemical compounds used in foodand flavor industries. Certain of the useful compounds derived from anincrease in the levels of CoA and A-CoA include, but are not limited to,succinate, isoamyl alcohol and isoamyl acetate. Esters such as isoamylacetate may be used in nail polish, lacquer coatings, plasticizers, foodflavoring compounds and other industrial applications. The increasedproduction of A-CoA is also useful in the production of target compoundssuch as esters, PHBs and polyketides.

[0028]FIG. 1 shows the intersection of metabolic pathways at the A-CoAnode. Pyruvate is oxidatively decarboxylated to A-CoA by pyruvatedehydrogenase (PDH), which subsequently enters the tricarboxylic acid(TCA) cycle. In the presence of an alcohol, A-CoA may be converted to anester using an alcohol acetyltransferase (AAT). In the presence ofinorganic phosphate (Pi), A-CoA may be converted to acetyl phosphate byphosphotransacetylase (PTA), which in turn may be converted to acetateusing acetate kinase (ACK).

[0029]FIG. 2 shows the involvement of pantothenate kinase (PanK) in theCoA biosynthetic pathway. Also shown is the negative regulation of PanKby CoA and acetyl CoA.

[0030] In general, the invention relies on the introduction of one ormore genes into a microorganism, which in turn result in increasedintracellular levels of CoA and/or A-CoA. In an embodiment of theinvention, an isolated recombinant construct comprising the geneencoding PanK is introduced into an E. coli strain.

[0031] In an alternate embodiment of the invention, an isolatedrecombinant construct comprising the gene encoding pyruvatedehydrogenase (PDH) is introduced into an E. coli strain together withan isolated recombinant construct comprising the gene encoding PanK.

[0032] In an embodiment of the invention, an E. coli strain istransformed with an isolated recombinant construct comprising the geneencoding PanK, where the panK gene is under the control of the lacpromoter and additionally comprising the ATF2 (Alcohol Acetyltransferase2) gene under the control of the ptb (Phosphotransbutyrylase) promoter.

[0033] In general, the invention relies on the introduction of one ormore genes into a microorganism, where the genes encode enzymes thatcatalyze one or more rate limiting steps of A-CoA biosynthesis. Anexample of an enzyme involved in a rate limiting step of A-CoA synthesisis pantothenate kinase. Overexpression of the gene encoding pantothenatekinase along with simultaneous supplementation of precursor pantothenicacid, significantly increases intracellular CoA levels (FIG. 1).

[0034] Another example of an enzyme involved in a rate limiting step ofA-CoA synthesis is pyruvate dehydrogenase. Overexpression of pyruvatedehydrogenase in the presence of elevated levels of pantothenate kinasealong with simultaneous supplementation of precursor pantothenic acid,leads to the increased carbon flux from pyruvate to A-CoA.

[0035] A third example of an enzyme involved in a rate limiting step ofA-CoA synthesis is pyruvate oxidoreductase. Overexpression of pyruvateoxidoreductase in the presence of elevated levels of pantothenate kinasealong with simultaneous supplementation of precursor pantothenic acid,leads to the increased carbon flux from pyruvate to A-CoA.

[0036] The inventive system and methods described herein may be used tomanipulate the production of A-CoA through the overexpression of anyactive enzyme that is capable of increasing the carbon flux through theA-CoA node.

[0037] An embodiment of the invention provides a method of increasingthe intracellular pool of A-CoA by elevated expression of at least onegene which encodes an enzyme involved in A-CoA biosynthesis.

[0038] As used herein, the enzymes involved in A-CoA metabolism includesall enzymes whose elevated expression results in an increase in thecarbon flux through the A-CoA node. These enzymes include enzymes thatmediate the conversion of pyruvate to A-CoA, as well as enzymes thatcatalyze one or more rate-limiting steps of the A-CoA biosynthesispathway. These enzymes include, but are not limited to, pyruvatedehydrogenase, pyruvate formate lyase, pyruvate oxidoreductase,pantothenate kinase, and mixtures thereof.

[0039] Another important enzyme that plays a role in the biosynthesis ofCoA is phosphopantetheine adenylyltransferase (CoAD). In an embodimentof the invention, overexpression of CoAD leads to the increased carbonflux through the A-CoA node.

[0040] In other embodiments of the inventions, the A-CoA level isenhanced through the deletion of an A-CoA utilizing pathway. Analternate embodiment of the invention shows an enhancement of A-CoAlevels through the reduction of A-CoA flux through one or more A-CoAutilizing pathways. Examples of such A-CoA utilizing pathways include,but are not limited to, acetate formation pathway of acetate kinase andphosphotransacetylase, the TCA cycle entry of citrate synthase (citratesynthase formation), the fatty acid biosynthesis pathway, the formationof malonyl-CoA (malonate formation), and the condensation of acetyl-CoAvia a thiolase (acetoacetate or acetoacetyl CoA formation). Thesestrategies for reduction of utilization of A-CoA can be used incombination with the strategies to increase acetyl-CoA to yieldadditional incremental increases that are useful in directing metabolismin particular types of cells. Additional ways to increase the level ofA-CoA directly through the enzymes that uptake acetic acid such as A-CoAsynthetase or other acyl-CoA synthetases that uptake other acids (e.g.,propionic acid or butyric acid) could be used in combination with theabove-listed strategies.

EXAMPLE 1 Plasmid Construction

[0041] Plasmid pGS367 (Pyruvate dehydrogenase expression plasmid) wasobtained from Dr J. R. Guest of Dept of Molecular Biology andBiotechnology, University of Sheffield, Sheffield, UK. Plasmid pSJ380bearing the panK (Pantothenate Kinase) gene cloned in pET-15b (NOVAGEN™)vector under the control of T7 promoter was obtained from Dr. SuzanneJackowski of Biochemistry Dept, St Jude Children's Research Hospital,Memphis, Tenn. A 1.5 kb XbaI-BamHI fragment containing the panK gene wascloned into the high copy number plasmid pUC19 to yield the constructpRV380, following which it was cloned into the plasmid pDHK29 using thesame restriction sites to yield the construct pRV480. The construct,pRV480, bearing the panK gene is compatible with pGS367. The ATF2(Alcohol Acetyltransferase 2) gene along with the ptb(Phosphotransbutyrylase) promoter was amplified by PCR the constructpTAAT (which carries the ATF2 gene of yeast) as template DNA. Theforward and reverse primers used were as follows:5′-CCCAAGCTTTGTGGATGGAGTTAAGTCAGTAGAAAG-3′ (forward primer); [SEQ ID NO:1] and 5′-CCATCGATTTAAAGCGACGCAAATTCGCC-3′ (reverse primer) [SEQ ID NO:2]

[0042] The forward and reverse primers contain HindIII and ClaIrestriction sites respectively, which allowed the amplified PCR fragmentto be cloned into the corresponding restriction sites of the plasmidpRV480 to yield pATCA (FIG. 3). The newly created pATCA constructcontains panK gene under the control of the lac promoter and ATF2 geneunder the control of the ptb promoter. This newly constructed plasmidpATCA, bearing the genes panK and ATF2 is compatible with pGS367.

[0043] Relevant plasmid constructs were transformed into DH10B or YBS121bacterial strain to carry out certain exemplary embodiments of theinvention.

[0044] The plasmids used in certain embodiments of the invention are setforth in Table 1 below. The transformed bacterial strains used incertain embodiments of the invention are set forth in Table 2 below.TABLE 1 Plasmid Properties pGS367 Pyruvate dehydrogenase expressionplasmid pRV480 Pantothenate kinase expression plasmid pATCA Pantothenatekinase expression plasmid where the panK gene is under the control ofthe lac promoter and additionally containing the ATF2 gene under thecontrol of the ptb promoter ptac-85 IPTG-inducible bacterial expressionvector

[0045] TABLE 2 ATCC Recombinant Deposit Strain No. PropertiesDH10B(ptac-85, Overexpresses pantothenate kinase pRV480) DH10B(pGS367,Overexpresses pantothenate kinase and pRV480) pyruvate dehydrogenaseDH10B(ptac-85, Overexpresses pantothenate kinase pATCA) expressionplasmid where the panK gene is under the control of the lac promoter andadditionally containing the ATF2 gene under the control of the ptbpromoter DH10B(pGS367, Overexpresses pantothenate kinase pATCA)expression plasmid where the panK gene is under the control of the lacpromoter and additionally containing the ATF2 gene under the control ofthe ptb promoter, and pyruvate dehydrogenase YBS121 Overexpressespantothenate kinase (pATCA, expression plasmid where the panK geneptac-85) is under the control of the lac promoter and additionallycontaining the ATF2 gene under the control of the ptb promoter YBS121Overexpresses pantothenate kinase (pATCA, expression plasmid where thepanK gene pGS367) is under the control of the lac promoter andadditionally containing the ATF2 gene under the control of the ptbpromoter, and pyruvate dehydrogenase DH10B(pUC19) Control DH10B(pRV380)Overexpresses panK DH10B(pKmAT, Control pUC19) DH10B(pKmAT,Overexpresses panK pRV380)

EXAMPLE 2 Bioreactor Experiments

[0046] Bioreactor studies were performed in a 1 liter (l) BIOFLO 110™fermentor with 0.5 l working volume to provide a controlled environmentwith 0.5 liter working volume. The dilution rate was maintained ateither 0.15/hr or 0.35/hr until it reached a steady state after 4 to 6residence times. The temperature was controlled at 37° C. The pH wasmeasured using a glass electrode (METTLER-TOLEDO™) and controlled at aset point of 7.0 by adding 3N HNO₃ or 3N NaOH. Dissolved oxygen (DO) wasmonitored using a polarographic oxygen electrode (METTLER-TOLEDO™) andthe DO was maintained above 80% saturation by an automated controllerwhich adjusts the agitation appropriately using a feed back controlloop. The air was filtered through a 0.22-μm inline filter and deliveredto the culture at a flow rate of 2.5 liters/min. The initial agitationspeed was set at 500 rpm. The effluent gases were bubbled through a 1 MCuSO₄ solution to prevent release of bacteria. Samples were taken duringthe steady state phase after 4, 5 and 6 residence times.

EXAMPLE 3 Aerobic Shake Flask Experiments

[0047] Since isoamyl alcohol and isoamyl acetate are volatile compounds,aerobic shake flask experiments were carried out in flasks capped withrubber stoppers. The rubber stopper facilitates headspace gas samplingfor analysis of volatile compounds (isoamyl acetate and isoamyl alcohol)and also prevents their escape from the flask. For aerobic cultures, 10ml culture medium was used in a 250 ml Erlenmeyer flask and preliminaryexperiments have shown that the high headspace to culture medium ratio(240:10 air-to-liquid ratio) provided sufficient aeration over thecourse of the experiment. The cultures were grown in an orbital shakerat the required temperature. At the end of the experiment (24 hrs), thecultures were analyzed for isoamyl acetate production.

EXAMPLE 4 Quantification of Isoamyl Compounds

[0048] Isoamyl alcohol and isoamyl acetate content was determined byheadspace gas chromatography. The flask or the tube, as the case may be,was heated at 50° C. for 30 minutes and 1 ml of head space gas wasinjected into HEWLETT-PACKARD™ 6000 series gas chromatograph equippedwith an ALLTECH™ 6′×¼″×2 mm POROPAK™ QS 80/100 column. A 6% ethylacetate solution was used as internal standard.

EXAMPLE 5 Acetate Formation in an Aerobic Chemostat

[0049] The specific acetate production rate for the two strainsDH10B(pUC19) and DH10B(pRV480) is shown in FIG. 4. The results show thatthe overexpression of PanK leads to an increase in acetate levels andsuggests that higher carbon flux through the A-CoA node was achieved byexpressing PanK. This result was confirmed by the decreased levels ofsuccinate in the strain expressing PanK (FIG. 4).

EXAMPLE 6 Overexpression of Pantothenate Kinase

[0050] The variation in CoA/A-CoA levels was studied in a batch reactorto study the overexpression of pantothenate kinase.

[0051] The intracellular CoA/A-CoA levels were studied using therecombinant strains DH10B(pUC19) and DH10B(pRV480) in a batch reactorusing M9 medium. The results show that the overexpression of PanK leadsto an increase in CoA/A-CoA levels (FIG. 5). Additionally, the increasein CoA levels is greater than the observed increase in A-CoA levels.

[0052] The intracellular CoA/A-CoA levels were studied in the same twostrains above in the presence of 5 mM pantothenic acid (FIG. 6a). Thestrain overexpressing PanK showed higher levels of intracellular A-CoAin the presence of pantothenic acid relative to the non-supplementedcontrol experiments.

EXAMPLE 7 Isoamyl Acetate Production

[0053] Two recombinant strains were constructed, DH10B(pKmAT, pUC19) andDH10B(pKmAT, pRV380). The latter strain overexpresses PanK and displayshigher isoamyl acetate production relative to the control strain (FIG.6).

EXAMPLE 8 Coa/A-Coa Levels

[0054] The variation in CoA/A-CoA levels was studied in an aerobicchemostat to study the coexpression of pyruvate dehydrogenase andpantothenate kinase, and the results are shown in FIG. 7. The precursorcompound pantothenic acid (5 mM) was supplemented in all theseexperiments as a substrate for the overexpressed pantothenate kinase toincrease intracellular CoA/A-CoA levels.

[0055] The intracellular CoA/A-CoA levels were studied using therecombinant strains DH10B(ptac-85, pRV480) and DH10B(pGS367, pRV480) inan aerobic chemostat using Luria Broth medium at two different dilutionrates (0.15/hr and 0.35/hr). Both strains overexpress pantothenatekinase and are supplemented with pantothenate in the culture medium,which enables them to have an elevated levels of intracellularCoA/A-CoA. However, only the strain DH10B(pGS367, pRV480) overexpressespyruvate dehydrogenase whereas the strain DH10B(ptac-85, pRV480) carriesa control plasmid. The intracellular levels of CoA/A-CoA are below thedetection limit of HPLC (˜0.04 nmol) for both the strains at a dilutionrate of 0.15/hr. At such a low dilution rate the E. coli culture atsteady state corresponds more to the stationary phase of cell growth.This observation is consistent with the observation that the CoA/A-CoAlevels were negligible in the stationary growth phase.

[0056] At a dilution rate of 0.35/hr, the intracellular CoA/A-CoA levelswere within the detectable range of HPLC. At this higher dilution rate,the cell culture at steady state corresponds to exponential growth phaseand the intracellular levels of CoA and A-CoA are significant anddetectable. This is again consistent with earlier studies where highlevels of CoA and A-CoA levels were observed during the exponentialgrowth phase. However, there was no significant change in theintracellular A-CoA level with the overexpression of pyruvatedehydrogenase in addition to pantothenate kinase (FIG. 7).

EXAMPLE 9 Glucose Uptake and Acetate Formation

[0057] The specific glucose uptake rate for the two strainsDH10B(ptac-85, pRV480) and DH10B(pGS367, pRV480) at two differentdilution rates is shown in FIG. 8. Both strains showed higher glucoseuptake rate at the higher dilution rates. At a dilution rate of 0.35/hr,the control strain DH10B(ptac-85, pRV480), exhibited a significantlyhigher uptake rate than DH10B(pGS367, pRV480), which overexpresses bothPanK and PDH.

[0058] The specific acetate production rate for DH10B(pGS367, pRV480) issignificantly higher than the control strain at both dilution rates(FIG. 9). At the dilution rate of 0.15/hour, DH10B(pGS367, pRV480)displays a 103% increase in acetate production. At a dilution rate of0.35/hour, DH10B(pGS367, pRV480) displays a 53% increase in acetateproduction. These results suggested that higher carbon flux through theA-CoA node was achieved by co-expressing both PanK and PDH.

EXAMPLE 10 Coexpression of PDH and Pank

[0059] Two recombinant strains were constructed, DH10B(ptac-85, pATCA)and DH10B(pGS367, pATCA). Both strains overexpress pantothenate kinasedue to which both strains have elevated CoA/A-CoA levels when the cellculture medium is supplemented with pantothenate. Similarly both thestrains overexpress alcohol acetyltransferase and therefore can produceisoamyl acetate when isoamyl alcohol is added externally to the cellculture medium. However, only the strain DH10B(pGS367, pATCA)overexpresses PDH thereby enhancing the carbon flux from pyruvate toA-CoA in this strain. The production of isoamyl acetate was studied inboth strains to elucidate the effect of this coexpression on isoamylacetate production. No increase in isoamyl acetate production wasobserved upon overexpression of pyruvate dehydrogenase in addition topantothenate kinase (data not shown).

[0060] The results of isoamyl acetate production can be explained if thecompetition of acetate production pathway at the A-CoA node is takeninto consideration. The enzyme alcohol acetyltransferase (AAT), whichcondenses isoamyl alcohol and A-CoA to form isoamyl acetate, might becompeting less effectively with phosphotransacetylase for the commonsubstrate A-CoA. Phosphotransacetylase (PTA) catalyses the formation ofacetyl phosphate from A-CoA, the first step in the formation of acetate.The PTA enzyme has greater affinity towards A-CoA when compared to AAT.This observation suggests that the acetate production pathway might bestronger than the ester production pathway and possibly drains theenhanced carbon flux.

EXAMPLE: 11 Channeling Enhanced Carbon Flux to Isoamyl AcetateProduction

[0061] Since the acetate production pathway is more competitive than theisoamyl acetate production pathway at the A-CoA node, it washypothesized that with the inactivation of acetate production pathway,the carbon flux could be more efficiently channeled to ester production.Under such conditions the enhanced carbon flux through the A-CoA nodecan have a beneficial effect on ester production. To test thishypothesis, a ackA-pta deletion mutant (a strain containing mutantcopies acetate kinase (ackA) and phosphoacetyltransferase (pta)) YBS121was used to construct two recombinant strains, YBS121(ptac-85, pATCA)and YBS121(pGS367, pATCA).

[0062] The supplementation of pantothenic acid is necessary in additionto overexpression of pantothenate kinase to increase intracellularCoA/A-CoA levels. This supplementation/non-supplementation ofpantothenic acid to the culture medium was used as control parameter tomaintain intracellular CoA/A-CoA levels at elevated/basal levels. Aseries of triplicate experiments were performed to study the effect ofCoA/A-CoA manipulation and PDH overexpression on isoamyl acetateproduction both individually and in combination. Even though the plasmidpATCA, overexpresses PanK, the supplementation of the precursorpantothenic acid is required to increase CoA/A-CoA levels. The resultsof these experiments are shown in FIG. 10.

[0063] The strain YBS121(ptac-85, pATCA) produced 0.07 mM isoamylacetate without supplementation of pantothenic acid. Uponsupplementation of pantothenic acid, the isoamyl acetate production inthe same strain increased to 0.16 mM, a 225% increase. These resultsindicate that the CoA/A-CoA manipulation leads to a 124% increase inisoamyl acetate production. However, the strain YBS121(pGS367, pATCA)produced 0.23 mM isoamyl acetate without supplementation of pantothenicacid, which is a 223% increase compared to the control strainYBS121(ptac-85, pATCA) (no pantothenic acid addition). This result showsthat overexpression of pyruvate dehydrogenase is more efficient inincreasing isoamyl acetate production compared to CoA/A-CoAmanipulation. However the same strain (YBS121(pGS367, pATCA)) produced0.44 mM of isoamyl acetate upon supplementation of pantothenic acid. Theincrease in isoamyl acetate production is about 5-fold, uponsimultaneous manipulation of CoA/A-CoA levels and enhancing carbon fluxfrom pyruvate node. This significant increase in isoamyl acetateproduction illustrate that the strategies of cofactor manipulation andcarbon flux enhancement are synergistic and much more effective inincreasing isoamyl acetate production, than using either of thestrategies alone.

EXAMPLE 12 PDH and Pank Coexpression

[0064] When the above experiments are repeated without anysupplementation of pantothenic acid, notable differences were observedin the accumulation of pyruvate and the results are as shown in FIG. 11.The ackA-pta mutation relieves the highly competitivephosphotransacetylase enzymatic step of the acetate formation pathwayand makes A-CoA more accessible to alcohol acetyltransferase. However,the inactivation of the acetate formation pathway leads to metabolicimbalance at the pyruvate node. The carbon flux is bottled up at thepyruvate node leading to excretion of pyruvate to the extracellularmedium. The recombinant strain, YBS121(ptac-85, pATCA), an acetatepathway deletion mutant strain, produced 13.69 mM of pyruvate asexpected. Increasing intracellular CoA/A-CoA levels increases thisexcretion slightly. When the intracellular CoA/A-CoA levels wereincreased in the strain YBS121(ptac-85, pATCA) upon pantothenic acidsupplementation, it produced 13.81 mM of pyruvate. Overexpression ofpyruvate dehydrogenase could convert some of this excess pyruvate toA-CoA leading to a decrease in pyruvate excretion. The strainYBS121(pGS367, pATCA), which overexpresses pyruvate dehydrogenaseproduced only 10.97 mM of pyruvate. This overexpression of pyruvatedehydrogenase lead to a 21% decrease in pyruvate accumulation. However,a significant amount of pyruvate is still excreted even in this case.The same strain YBS121(pGS367, pATCA) when supplemented with pantothenicacid, produced only 1.1 mM of pyruvate, which is a significant drop inpyruvate excretion, when compared to the control strain YBS121(ptac-85,pATCA). When the overexpression of pyruvate dehydrogenase is accompaniedby an increase in availability of CoA, most of the excess pyruvate couldbe efficiently converted to A-CoA. The coexpression of pyruvatedehydrogenase and pantothenate kinase relieved the metabolic imbalanceat pyruvate node and the pyruvate excretion dropped to negligiblelevels. This metabolic engineering strategy efficiently channels theexcess carbon flux from pyruvate node to A-CoA node in an acetatepathway deletion mutant. The drop in pyruvate excretion leads to a moreefficient utilization of the carbon source without any loss at thepyruvate node.

1 2 1 36 DNA Artificial primer 1 cccaagcttt gtggatggag ttaagtcagt agaaag36 2 29 DNA Artificial primer 2 ccatcgattt aaagcgacgc aaattcgcc 29

What is claimed is:
 1. A method of manipulating the metabolism of acell, comprising elevated expression of one or more enzymes involved inA-CoA metabolism, wherein said one or more enzymes are involved in oneor more rate limiting steps of A-CoA synthesis.
 2. The method of claim1, wherein the enzymes are selected from the group consisting ofpyruvate dehydrogenase, pyruvate formate lyase, pyruvate oxidoreductase,pantothenate kinase, phosphopantetheine adenylyltransferase andcombinations thereof.
 3. The method of claim 2, where the cell expressesone of the group consisting of i) overexpresses pantothenate kinase; ii)overexpresses pantothenate kinase and pyruvate dehydrogenase; iii)overexpresses pantothenate kinase where the panK gene is under thecontrol of the lac promoter and additionally overexpresses the ATF2 geneunder the control of the ptb promoter; and iii) overexpressespantothenate kinase expression plasmid where the panK gene is under thecontrol of the lac promoter and additionally overexpressing the ATF2gene under the control of the ptb promoter, and pyruvate dehydrogenase.4. A method of increasing the A-CoA flux in a cell comprising elevatedexpression of one or more enzymes involved in A-CoA metabolism, whereinsaid one or more enzymes are involved in one or more rate limiting stepsof A-CoA synthesis.
 5. The method of claim 4, wherein the enzymes areselected from the group consisting of pyruvate dehydrogenase, pyruvateformate lyase, pyruvate oxidoreductase, pantothenate kinase,phosphopantetheine adenylyltransferase and combinations thereof.
 6. Amethod of manipulating the metabolism of a cell, comprising deletion ofone or more A-CoA utilizing pathways.
 7. The method of claim 6, whereinsaid one or more A-CoA utilizing pathways are selected from the groupconsisting of acetate formation pathway, citrate synthase formationpathway, fatty acid biosynthesis pathway, malonate formation pathway,and acetoacetate formation pathway.
 8. A method of increasing the A-CoApools in a cell comprising deletion of one or more A-CoA utilizingpathways.
 9. The method of claim 8, wherein said one or more A-CoAutilizing pathways are selected from the group consisting of acetateformation pathway, citrate synthase formation pathway, fatty acidbiosynthesis pathway, malonate formation pathway, and acetoacetateformation pathway.
 10. A method for the biosynthesis of one or moretarget compounds comprising increasing the intracellular levels of A-CoAand directing the increased A-CoA levels towards the biosynthesis ofsaid one or more target compounds.
 11. The method of claim 10, whereinthe intracellular levels of A-CoA are increased by elevated expressionof one or more enzymes involved in A-CoA metabolism.
 12. The method ofclaim 10, wherein the intracellular levels of A-CoA are increased bydeletion of one or more A-CoA utilizing pathways.
 13. The method ofclaim 10 wherein said one or more target compounds are selected from thegroup consisting of succinate, isoamyl alcohol, isoamyl acetate, esters,PHBs and polyketides.
 14. A method of producing isoamyl acetate in acell comprising expressing at elevated levels one or more enzymesinvolved in A-CoA metabolism, wherein said cell displays increased fluxthrough the A-CoA node.
 15. The method of claim 14 wherein said one ormore enzymes are involved in one or more rate limiting steps of A-CoAsynthesis.
 16. The method of claim 15, wherein the one or more enzymesare selected from the group consisting of pyruvate dehydrogenase,pyruvate formate lyase, pyruvate oxidoreductase, pantothenate kinase,phosphopantetheine adenylyltransferase and combinations thereof.
 17. Amicroorganism which expresses one or more enzymes involved in A-CoAmetabolism at elevated levels, wherein said microorganism displaysincreased flux through the A-CoA node.
 18. The microorganism of claim17, wherein said one or more enzymes are involved in one or more ratelimiting steps of A-CoA synthesis.
 19. The microorganism of claim 18,wherein the one or more enzymes are selected from the group consistingof pyruvate dehydrogenase, pyruvate formate lyase, pyruvateoxidoreductase, pantothenate kinase, phosphopantetheineadenylyltransferase and combinations thereof.
 20. The microorganism ofclaim 17, wherein said microorganism is selected from the goupconsisting of ATCC ______, ______, ______, ______, ______, ______.
 21. Amethod of increasing CoA pools, comprising producing increased levels ofpantothenate kinase (PanK) activity in a cell together with providingincreased pantothenic acid levels, sufficient to increase the pool ofCoA in the cell.
 22. The method of claim 21, wherein producing increasedlevels of PanK activity is achieved by transforming the cell with avector that overexpresses the PanK gene and increased pantothenic acidis provided in a medium used to grow the cells.
 23. The method of claim21, wherein producing increased levels of PanK activity is achieved bymanipulating the host genome to overexpress the PanK gene and increasedpantothenic acid is provided in the cell medium.
 24. A method ofincreasing synthesis of CoA containing compounds from a bacterial cell,comprising producing increased levels of pantothenate kinase (PanK)activity in a cell together with providing increased pantothenic acidlevels, sufficient to increase the pool of CoA in the cell and drive thesynthesis of CoA containing compounds.
 25. The method of claim 24,wherein producing increased levels of PanK activity is achieved bytransforming the cell with a vector that overexpresses the PanK gene andincreased pantothenic acid is provided in a medium used to grow thecells.
 26. The method of claim 24, wherein producing increased levels ofPanK activity is achieved by manipulating the host genome tooverexpresses the PanK gene and increased pantothenic acid is providedin the cell medium.